Preparation of 2-keto gulonic acid and its salts



Junes, 1947. B GRAY 2,421,612

PREPARATION OF 2-KETO GULONIC ACID AND ITS SAIAJISl Filed lay 4, 1945 l and d-gluonafe 02 0r H2604 calcium l-idanafe Oxiddl'ive -fermenfal'lon E o o in Juifable culture medium 25 -90 C- wim film forming bacteria,

' e. g. Acefobacler ``uboxydaf1.s

, 'nfco 1 0 o Y calcium 5-kefo gprm H O H H O H l gluconclfe ammonium H0 `M Ca H0 H Ca Saparal'ian sul? l'l` Hg ZH Recyclm-" H2 o H H2 o H calcium lzz.Acidify 2 2 l-idonae W- calcurn 5keo calcium Y glucona'l-e l-idonae (insoluble) (soluble) Y l-ldonic 1acid+Ca`04 EZ' l Fille l-donic acid solulion ZX` l pimeriza'lion agrmf Puri-fcaTlon and A efgfdl' calcium 1 Isolafion of Coo H u oh l g I aTe l-gulomc acid 1 H0 I H as calcium sal-I' H9 H 1 Y H (|30 H Oxdalive -fermenfal'ion in suifable H 0 H` cullare medium, wlfhfilm j c Hz 0H 1 X formln ac l l lag.` Ace'robacl'er uboxydans P9 ""C Cid luricl'ianqal l-donc acid l l 3mm.;

,Ho H Ca H gc H LIyrpnL'. Gray, H110 H p the said l-gulonic acid or its Patented June `3, 1947 PREPARATION F Z-KETO GULGNIC ACID` l ANDITS SALTS Byron E. Gray, Alton, Ill. t Application May 4, 1945, Serial No. 591,879

This invention relates to a new and improved` method for the manufacture of 2keto`gulonic It is Well known inthe art that Z-keto-l-gulonid acid, or its esters, serves as a ilnal intermediate for the manufacture of the physiologically im-v portant l-ascorbic acid, better known as synthetic vitamin C. 1.

The established conventional processv for producing Z-keto-l-gulonic acid isv rather complicated, consisting of reduction :of d-glucose to sorbitol, bacterial oxidation of sorbitol to sorbose,

formation of di-acetonejso'rbose, oxidation of diacetone sorbose to di-acetone 2-keto-l-gulonic acid, and hydrolysis of` di-acetone 2-keto-lgulonic acid, to give Z-keto-l-gulonic acid.-

(Reichstein and, Gruessner, Helv. Chim, Acta, 17:311,1934.) 4

A principal object of this invention is the provision of anew method for the preparation of 4 Claims. (Cl. 19d-47) rend., 102:924 and 1l1:185;" Stubbs et al.. Ind.

1940)` The -ketof` gluconic acid is usually isolated as the 'almost in soluble calcium salt, containing `2%. molecules of and Eng. Chem., 32:1626,

water of crystallization.

It is known that the calcium' salt of dinuconic acid may be converted by oxidative fermentation to the almost insoluble calcium salt of y5keto` Ind. and` Eng; Chem., 32:1626, 1940.) Said oxidative fermentaf gluconic. acid. t (Stubbs et al.;l

tion d oes not ailect the salts of l-idonic acid.

It is also known that the calcium salt of 5f-keto` gluconic acid may be hydrogenated in thepresenceof a suitable catalyst to give amixture of.`

calcium l-idonate and calcium d-gluconate.` (Pasternack and Brown, U. S. Patent, 2,168,878.)

I have now foundthat the ammoniumiras wen v as the alkali metal salts, e. g., `potassium and so -keto-l-gulonic acid or its salts.l A further obi ject is the provision "of a new procedure forthe production of 2-keto-l-gulonic-acid or its salts from l-gulonic acid and its salts. Still further.

objects include the provisionof a new procedure for the production of vitamin C intermediate from d-glucose.` and, ln turn, 5ketodgluconic acid witha high degree of efiiciency and resulting good yields and the provision of a newmethod for the `production of l-idonic acid from 5-keto- .d-gluconic acid by hydrogenation.

'I'hese objects are accomplished according to the process of my invention by hydrogenating the ammonium salt of 5-keto gluconic acid to obtain a mixture of ammonium d-'gluconate and ammonium 1-idonate, converting 'the said mixture into the corresponding calcium saltsl of l-idonic and d-gluconic acid, eliminating the calcium salt of d-gluconic acid from the mixture of oxidative fermentation as the insolubleI calcium salt of 5-keto gluconic acid, epimerizing the resultant remaining l-idonic acid or its salts to give l-gulonic acid, its saltsv or lactone, and converting salts to Z-keto-lgulonic acid or its salts by oxidative fermentation.` i

Discussion or rm: Paocass Reference is made" to the attached drawing which constitutes anzabbrevated flow-sheet of my process, in order to aid in a clearer understanding of this invention.

(a) Preparation of l-idonic acid and/or its salts It has been found that `S-keto gluconic acid dium) salt of said -ketogluconic 'acid iscapable of hydrogenation in substantially the same manner as the calcium salt, but, hydrogenation is easier and lower pressures or `temperaturesmay be used if desired with these more soluble salts.`

The mixture of ammonium salts of lfidonic acid and d-gluconic acid resulting from said hydrogenation of the ammonium-salt of 5keto gluconic acid (step I) kis treated with-lime (calcium hydroxide.) and heatedfwhich drives the ammonia oifand gives a mixtureof the calcium salts" of l-idonic and d-gluconicacid (step II). Excess calcium hydroxide in said mixture is suitably neutralized (step III), for `example with` carbon di-` oxide or sulfuric acid. and the resultant calcium carbonate or sulfate iiltered oiT. leaving a neutral solution of calcium l-idonate and d-gluconate;

To this solution is now-added d-glucose (corn` sugar), suitable nutrients, and a buffering agent,

- such as calcium carbonate, and the resultant culv ture liquid is fermented (step IV) with bacteria Y of the Acetobacter group (or other illm forming bacteria) Such fermentation may be done either bythe shallow pan surface growth method, without aeration, or by submerged growth, maintaining` the culture liquid in a high state of agitation, with simultaneous introduction of large quantities of air, .either under atmospheric or supery atmospheric pressure. Yields and rate of oxidative fermentation are increased "by' the latter method. `The culture medium" may be keptat may be easily `produced from the readily available d-glucose (corn sugar) either by chemical oxidation (Kiliani,` Berichte, 55Bz2817, 1922) or by oxidative fermentation. (Boutroux, Compt.

various temperatures, but the best range is 25- 30C. i V `1^ When oxidative fermentation is complete,the calcium, d-gluconate, together with most of the d-glucose not consumed by the bacteria, will be found to have been converted to calcium i-keto gluconate. Said calcium V5-keto gluconate, being almost insoluble, may be ltered oil, leaving the t unchanged calcium l-idonate in solution (stepN).

The calcium 5-keto gluconate thus obtained asa by-product may be used as a raw material for the filtered off.

thus obtained is Y contains l-gulonicacid. Itis to a syrup; whereupon l-gulonic acid (step X).

ammonium -keto gluconate in the previously de'- scribed hydrogenation (step XI) i (b) Preparation of l-yillomc acid and/or its salts or lactone y VII).

'Iofthe filtrate, consisting of an aqueous solution of l-idonic4 acid, is added pyridine, solution is heated atl 140 C. under pressure for three hours, or boiled under reflux `for. 115 hours (step VIII). Calcium and the pyridine distilled offY and recovered. The

then filtered, eliminating excess calcium hydroxide, and evaporated to dryness.

To the dry residue of crude calcium l-gulonate and the then are capable of oxidizing the l-gulonic acid to the corresponding 2-keto acid.

Such oxidative fermentation of the above-described culture solution may be carried out by the surface growth shallow pan method, without,V aeration, or by submerged growth,'maintaining 1 Y the solution in a 1 'gh state of agitation, with simultaneous introduction of large quantities of air, either under atmospheric or super-atmospheric pressure. Yields and rateV of oxidative fermentation. are increased The culture medium maybe kept at various tem-l peratures, but the optimum range is -30 C.

' the salt may hydroxide is then added.

When oxidative action is complete, the culture solution is clarified, filtered, concentrated under reducedpressure and cooled, whereupon'the calcium salt ofV l-xylo 2-keto hexonic acidcrystallizes and is filtered off and dried. Alternately,V

centrated solution with alcohol. *The calcium l-xylo 2-keto hexonate is easily changed into the y free acid by treatment with sulfuric acid, be converted directly into an ester by treatment with acid in anhydrous alcohol solution.

and unchanged l-idonate' is addedhydrochloric 'acid and benzaldehyde, withvigorous agitation. 'Ilhe insoluble di-benzal derivative of l-ldonic acid soon separates and, after dilution with water, is The di-benzall-idonic acid is easily hydrolyzed .to benzaldehyde and l-idonic acid with the subsequent batch as described above.

The nitrate, from the di-benzai l-idonid Vacid carefully'neutralized with calcium carnate, and. anequal volume of alcohol is added. Calcium l-guonate separates and is filtered off (step 1X). It may be puriiled by recrystallization from water, or by converting to the lactone `thru treatmentwith sulfuric acid. to remove the calcium ion, filtering oir tl'ie resultant calcium sulfate, and concentrating the lactone separates on standing. The lactone is easily converted into calcium 'l-gulonate, by heating with calcium carbonate.

y (e) Preparation of aceto-aguante acid (z-ylo- It has been found that certain bacteria ofl the Acetobacter series (as well as other species of illm forming bacteria) have the power to oxidize polyhydric alcohols possessing the proper configuration to corresponding keto alcohols. (Hann etal.,

J. Am. Chem. Soc, 6011201, 1938; Bertrand, Ann.

' chim. phys., 3:181, 1904.) The factors pertaining to such oxidation are summed up by Fulmer et al..

Iowa State College Journal of Science, 15:30, A

- g1. have now V`*found that this type of bacteria alsohas the power to .oxidizegulonic acid, to the corresponding 2-keto acid. Such bacteria have the power to produce enzymes capable of oxidizing the gulonic acid, but cannot well assimilate the acids. However, if a salt (for example, calcium) of l-gulonic acid is oxldatively fermented by a member of the Acetobacter or other film kforming series of bacteria, 1in the presence of a small amount of suitable substrate such as glycerol, corn sugar, mannitol. sorbitol, or maltose, there is formed the corresponding salt of 2-keto- 4the bacteria to grow and produce enzymes which The substrate permitsY quired amount of water.

The operation'of my process may be prehended from theV following plein whichall parts are byweight unless otherwisespecied.

fully com- A suspension of 172 partsof calcium l5keto gluconate, 21/2H20 in 2000 partsof distilled Water is prepared The suspension is cooled to 20 C. and sulfuric acid'carefully added with agitation until all the 5-ke'to gluconic acid is set free. Temperatureis kept at 20 C. or below during addition ofY acid. Y

' justed to 6.0 plus or minus 0.1.

' consisting of an Vheated to 50 let. vThe solution Vare added. Y The "Acetobacter suboxydans grown The mixture is next carefully neutralized to litmus with ammonia, gaseous or aqua. It is then C. and filtered. To the filtered solution of ammonium v5-keto gluconate are added 25 parts ofRaney nickel catalyst and the f mixture is placed in an agitated autoclave and heated at 60 C. under a hydrogen pressure ofY atmospheres. Progress of hydrogenation may be followed lby testing with Fehlings solution.

Ordinarily,` the hydrogenation will take about four or six hours.-

, When hydrogenation is complete, the pressure l is released and the catalyst is filtered off. Catalyst may be reused if the hydrogen used was pure. If not, it m'ay be regenerated. The filtrate is treated with 75 parts of hydrated lime and heated until substantially al1 the ammonia has Vbeen driven off. The mixture is then cooled and neutralized with carbon dioxide, a little activated carbon is added, and the solution is filtered and concentrated to '1000 parts.

To the filtered and clarified solution are added 75 parts glucose, 0.3 part octadecyl alcohol, and the pH -is Vad- 'Ihe solution is now placed in a suitable vessel equipped with a distributor inlet for sterile air alundum ball. The vessel also has an air outlet, and a sample withdrawal outis sterilized by heating at 15 lbs. I for l5 minutes, cooled, andV 11 parts calcium carbonate (sterilized separately)V culture solution oculatedwith 50 parts of a 48 on a 5 per cent sorbitol-0.5 per cent yeast extract liquid tube culture, and the inoculated medium is agitated Y vigorously with sterile air for a period of 8 days.

After the fermentation is complete, the precip- 1 be' precipitated from the cool conillustrative exam'- by agitation of the salt inthe re- 6 parts corn steeping liquor, and y is then in- Y hour culture of About 1'75 parts are obtained,

in place and the mixture heated on the oilbath at 140 C. for three hours.

At the end of three hours, the solution is diluted with 100 parts' water, 20 lparts of calcium hydroxide are added, and the mixture is heated until all the pyridine has been distilled off and recovered. The mixtureis next filtered to remove excess calcium hydroxide, and evaporated to dryness.

To the residue from the evaporation are next added 40 parts benzaldehyde and 80 parts of hydrochloric acid (sp. gr. 1.19) with vigorous agitation. Presently, a. precipitate of di-benzal l-idonic acid separates and after dilution with a little water is filtered off. About 60 parts are obtained. The filtrate contains l-gulonic acid. Calcium carbonate is now carefully added until the mixture is neutral to litmus. To the mixture is now added an equal volume of alcohol. The calcium l-gulonate precipitates and is filtered of. It is purified by dissolving in the minimum amount of hot water, adding a little carbon and filtering, cooling, andl reprecipitating with alcohol. Yield is 15 parts of the calcium salt of l-gulonic acid.

The di-benzal l-idonic acid is reworked by suspending it in ten times its weight of a 50% alcohol-50% N/10 sulfuric acid mixture and heating under reflux for one hour. 'I'he mixture is then neutralized to litmus with calcium carbonate and the separated mixture of calcium lidonate and calcium sulfate is filtered off. The l mixture may be added to the subsequent batch of calcium l-idonate before epimerization.

120 parts of puried calcium l-gulonate, obtained as previously described., are dissolved in water and the solution is made up to 1000 parts. To this solution are now added 5 parts corn steeping liquor, 0.3 part octadecyl alcohol, 5 parts maltose or sorbitol, and the pH is adjusted to 6.0 plus or minus 0.1.

The solution is now charged into a fermentation vessel and sterilized at 15 lbs. steam pressure for 15 minutes. which it is inoculated with 50 parts of the Acetobacter suboxydans culture indicated above. The inoculated medium is aerated vigorously, using a porous alundum ball, for 8 days. Course of fermentation may be followed by testing with Fehlings solution.

At the end of the fermentation, a little activated carbon is added to the solution, and is filtered. It is then concentrated under to a syrup (100-200 parts), cooled, and two volumes of ethyl alcohol are added. The calcium 2-keto-l-gulonate separates and is filtered ofi and dried. Yield is parts.

Having thus described my invention, what I f claim for Letters Patent is:

I claim:

l. The process for the production of a material from the group consisting .of 2-keto gulonic acid and its calcium salt, which comprises oxidative.

fermentation of a substance from the group consisting of gulonic acid and its presence of the bacteria Acetobacter suboxydans.

2. The process of claim 1, wherein the fermen- 1 tation is conducted by cultivating vthe bacteria in a submerged state, with aeratio 3. The process of claim l, wherein the fermentation is conducted by cultivating the bacteria in surface growth.

4. The process for the production of a material fromV the group consistingof 2`keto gulonic acid and its calcium salt, which comprises inoculating an aqueous solutionof the calcium salt of gulonic acid with suboydans and oxidatively fermenting said inoculated solution. i

BYRON E. GRAY.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS OTHER REFERENCES Fischer & Stahel, Berichte 24, 529 (1891). Stubbs et al., Ind. Eng. Chem. 32, 1626 (1940).

It is then cooled to 25 C. after reduced pressure calcium salt in the the bacteria Acetobacter` 

